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hecklist, diversity and distribution of testate amoebae in Chile

eonardo D. Fernándeza,b,c,∗, Enrique Laraa, Edward A.D. Mitchella,d

Laboratory of Soil Biology, Institute of Biology, University of Neuchâtel, Rue Emile Argand 11, CH-2000, Neuchâtel, SwitzerlandLaboratorio de Ecología Evolutiva y Filoinformática, Departamento de Zoología, Facultad de Ciencias Naturales y Oceanográficas,niversidad de Concepción, Barrio Universitario s/n, Casilla 160-C, Concepción, Chile

Centro de Estudios en Biodiversidad (CEBCH), Magallanes 1979, Osorno, ChileBotanical Garden of Neuchâtel, Chemin du Perthuis-du-Sault 58, CH-2000 Neuchâtel, Switzerland

bstract

Bringing together more than 170 years of data, this study represents the first attempt to construct a species checklist andnalyze the diversity and distribution of testate amoebae in Chile, a country that encompasses the southwestern region of Southmerica, countless islands and part of the Antarctic. In Chile, known diversity includes 416 testate amoeba taxa (64 genera,52 infrageneric taxa), 24 of which are here reported for the first time. Species−accumulation plots show that in Chile, theumber of testate amoeba species reported has been continually increasing since the mid-19th century without leveling off.estate amoebae have been recorded in 37 different habitats, though they are more diverse in peatlands and rainforest soils.nly 11% of species are widespread in continental Chile, while the remaining 89% of the species exhibit medium or short

atitudinal distribution ranges. Also, species composition of insular Chile and the Chilean Antarctic territory is a depauperatedubset of that found in continental Chile. Nearly, the 10% of the species reported here are endemic to Chile and many of themre distributed only within the so-called Chilean biodiversity hotspot (ca. 25◦ S-47◦ S). These findings are here thoroughly

Published in European Journal of Protistology 51, issue 5, 409-424, 2015 which should be used for any reference to this work 1

iscussed in a biogeographical and evolutionary context.

; South

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eywords: Amoebozoa; Biogeography; Ecology; SAR supergroup

ntroduction

Testate amoebae are a polyphyletic group of shelled pro-

ists present in many terrestrial, freshwater and occasionally

arine habitats. Their sensitivity to slight changes in the envi-onmental conditions has turned them into an increasingly

∗Corresponding author at: Laboratory of Soil Biology, Institute of Biol-gy, University of Neuchâtel, Rue Emile Argand 11, 2000 Neuchâtel,witzerland.

E-mail addresses: limnoleo@gmail.com, leonardo.fernandez@unine.ch L.D. Fernández).

2 e H c a mPab

America; The Antarctic

sed group of bioindicators to monitor pollution (Nguyen-iet et al., 2007; Meyer et al. 2012), ecosystem restoration

nd management (Turner and Swindles 2012; Valentine et al.013), cadaver decomposition (Seppey et al. 2015; Szeleczt al., 2013) or natural ecological gradients (Opravilova andajek 2006; Payne 2011; Koenig et al. 2015). Their shells

an be preserved for a long time in peats and sediments,nd ancient communities are nowadays commonly used to

onitor past environmental changes (Mitchell et al. 2008; ayne and Pates 2009). To date, testate amoebae have been ssigned to two different deep eukaryotic lineages: Amoe-ozoa (lobose testate amoebae) and the supergroup SAR

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filose testate amoebae) (Adl et al. 2012). Past estimationsf their diversity reached about 2,000 species (Meisterfeld002a, 2002b). However, these estimations are currentlyeing revised due to the introduction of DNA barcoding,hich revealed an important cryptic diversity (Kosakyan et

l. 2012; Heger et al. 2013; Singer et al. 2015). Notably, itas been shown in several cases that allopatric specia-tionccurred, and several species are known to have limitedeographic distributions as demonstrated with both morpho-ogical observations (Smith and Wilkinson 2007; Smith etl. 2008; Zapata and Fernández 2008) and molecularethods (Heger et al. 2013).The study of the diversity and distribution of testate

moe-bae boasts a long tradition (Penard 1902; Claparèdend Lachmann 1868; Ehrenberg 1853; Leidy, 1879).owever, most studies on these microorganisms have beeneveloped in the Holarctic region (Foissner 1997, 1999;mith et al. 2008). Data from the Southern Hemisphere aretill rare and sparse, indicating a limited knowledge on thesero-tists along a considerable fraction of the terrestrialurface of the planet. Chile encompasses almost all theouthwest-ern region of South America, countless islandsnd part of the Antarctic continent and covers a vast array oflimatic zones. Therefore, this country has a considerablenterest for testate amoebae biogeography. Despite a longistory of records of testate amoebae from Chile, the datare still scat-tered and incomplete. Knowledge of Chileanestate amoebae began with Ehrenberg (1843), who studied few samples col-lected at Cape Horn (Horn Island, Chile),he southernmost headland of Tierra del Fuego. Forty-sixears later, Certes (1889) reported several new species andenera after studying samples collected from 1882 to 1883y the French Scien-tific Mission to Cape Horn at Orangeay (Hoste Island, Chilean part of Tierra del Fuego). In thisanuscript, Certes described remarkable species such aspodera vas (Certes, 1889) and Certesella martiali (Certes,889). Subsequently, Wailes (1913) added some newecords and species from samples collected in Punta Arenas,alparaíso and Antofa-gasta from austral, central andorthern continental Chile, respectively. It is noteworthyhat back then, all mentioned localities were importanteaports, which were (or still are) part of internationalommercial sea routes. This probably largely determinedhe selection of sampling sites at that time. Later, Jung1942a) published a paper documenting a large number ofnown and then unknown taxa that he observed in samplesollected both in the Reloncaví Inlet and the inland territoryf south-central Chile. This paper was important for theatural history of Chilean testate amoe-bae as it broke therend of sampling exclusively at seaports and documentedhe species that were present in remote and often pristineites. Some years later, Hoogenraad and de Groot (1951)

eported some testate amoeba species that they found in oss samples collected in austral and cen-tral Chile. Then, onnet (1966) investigated the community composition and istribution of testate amoebae in south-central Chile. He escribed some new species and found

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hat the distribution of these organisms was mainly deter-ined by the C:N ratio, pH and water availability in

ifferent terrestrial habitats. More recently, Smith (1978,985) docu-mented the testate amoeba species compositionf the Chilean Antarctic territory, revealing a pauperizediversity. Like-wise, Zapata and colleagues furtherocumented the testate amoeba species composition ofouth-Central Chile (Zapata and Rudolph 1986), the Chileanntarctic territory (Zapata and Matamala 1987) and Easter

sland (Zapata and Crespo 1990). Finally, the 20th centurynded with the contribution of Golemansky and Todorov1996), who described for the first time the psammobioticauna from the marine littoral and supralittoral of Chile. Theurrent century has witnessed an increase in the number oftudies involving testate amoebae. Several papers describinghe Chilean testate amoebae fauna and ecology as well asroviding new records or describ-ing new species have beenublished during the last years (Zapata et al. 2002; Zapata005; Zapata et al. 2007a, 2007b; Zapata and Fernández008; Zapata et al. 2008; De Smet and Gibson 2009;ernández and Zapata 2011; Santiba˜nez et al. 2011;ernández et al. 2012; Chatelain et al. 2013; Fernández015).

Bringing together more than 170 years of data accumu-ated by numerous researchers, this paper represents the firstttempt to synthesize the knowledge about Chilean testatemoebae, including both published and unpublished informa-ion. The first purpose of this study was to give an updated andomprehensive species checklist that includes nomenclaturehanges prompted by recent research as well as ecologicalnformation and geographical data on the distribution of eachpecies in the whole Chilean territory including continentalhile, insular Chile and the Chilean Antarctic territory. The

econd purpose of this study was to investigate the diver-ity and spatial patterns of these organisms in Chile usingualitative and quantitative approaches including estimationsf richness at different taxonomic levels, habitats surveyed,abitat preference, latitudinal patterns of distribution, levelsf endemicity and comparisons among the species composi-ion recorded on insular, Antarctic and continental Chile. Wexpect that this manuscript will contribute to reduce the exist-ng knowledge gap between South American and Holarcticestate amoebae and encourage the local research interest inhis group.

aterial and Methods

eographical setting

Chile is a long and narrow country situated on the Pacificoast of South America, stretching over 4300 km southwardsrom latitude 17◦30′ S to Cape Horn at 56◦ S. Chile also has

2

overeignty over the Pacific islands of Easter Island and theuan Fernández Archipelago and claims Antarctic Territoryetween 53◦ and 90◦ W. As a result, Chile is usually parti-ioned in three political divisions: continental Chile, insular

Fig. 1. The Chilean territory includes: (a) continental Chile, whichstretches from 17◦30′ S to Cape Horn at 56◦ S; (b) insular Chile,which includes many continental and oceanic islands (the latterare indicated by the dotted circle); and (c) the Antarctic territoryincluded between 53◦ W and 90◦ W.

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cawipmAlong with the record of each taxon, we also took note of: (a)its species authority; (b) synonyms used in the Chilean testate

hile and the Chilean Antarctic territory (Fig. 1). Chile isegarded as a land of climatic and geographic extremes.oughly, the climate of continental Chile is dry and hot in

he north, mostly Mediterranean in the central region, andoth subpolar and oceanic in the southern region. The cli-ate of insular Chile is either subtropical or oceanic and thehilean Antarctic territory is dominated by a polar climate.hus, Chile overall includes many major biomes includ-ingot and cold deserts, alpine, arid shrublands, deciduousorests, rainforests and grasslands. A considerable part ofhe Chilean territory also represents a biodiversity hotspotalled the Chilean Winter Rainfall-Valdivian Forestsotspot (i.e. the Chilean hotspot) (Arroyo et al. 2004). Thehilean hotspot stretches between 25◦ S and 47◦ S,

ncluding a nar-row coastal strip between 19◦ S and 25◦ S,

lus the Juan Fernández Archipelago. a

atabase constructionTo construct the database used in this study, we first did

n exhaustive literature search that included all publishedaterial devoted, partially or completely, to testate amoebae

f Chile. Second, we performed a comprehensive review ofaterial stored both in the private collection of Jaimeapata (a retired Chilean protistologist) and in the protistollec-tion of the Universidad de Los Lagos (Chile). Thisaterial consisted altogether of 250 permanent slides and

00 dry moss samples of 5−10 g each collected between000 and 2008 all over continental Chile. We examinedndividually each permanent slide to ensure that all specieseported had been properly identified. For moss samples, weook a sub-sample of 3 g of dry moss, re-hydrated it in 250l of distilled water and sieved it through 400 and 15 �mesh sieves. Each fraction was examined under an invertedicroscope for documenting species occurrences. The

dentification of taxa was based on morphological charactersf the shell fol-lowing traditional taxonomic references.mportant sources used in the identification of amoebaencluded those by Certes (1889); Jung (1942a, 1942b);

azei and Tsyganov (2006) and Zapata et al. (2007a,007b). When possible, the amoebae observed werehotographed by means of a digital cam-era connected to annverted microscope and/or by scanning electronicroscopy (SEM). For SEM microscopy, the indi-vidualsere cleaned by several transfers through distilled water,ounted on stubs and finally air-dried. The individ-uals on

tubs were coated with gold and photographed with either aEOL JSM-6380 or Philips XL30 operating between 10 and5 kV. Third and finally, several species identified fromamples collected by the first author between 2007 and 2013n insular and continental Chile (6275 soil/moss/litteramples of ca. 10 cm3 each) were also integrated into theaw database. The analyses of these samples as well as theso-lation and identification of these species followed theame process described above for dry moss samples.

As we compiled data from various sources that were gen-rated using different methods and taxonomical approaches,e needed to correct several spelling errors, misidentifica-

ions and obsolete taxon names (e.g. synonyms, homonymsnd nomina nuda taxa). We also sent our raw database toolleagues who also have taxonomic expertise in the groupo have their opinion (see acknowledgments section). Theiromments and suggestions were taken into account in thenal version of the database.We thus obtained a comprehensive and updated species

hecklist for the Chilean testate amoebae. Taxa identifiedt species level included forms indicated as confer (cf.) orith a question mark (?), meaning that organisms were sim-

lar to described species but not identical, thus representingrobably new species. Infraspecific taxa included those taxaentioned as subspecies, varieties or forms in the literature.

3

moebae literature; (c) infraspecific taxa used in the Chilean

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iterature; (d) the habitat(s) where it has been recorded inhile; (e) additional notes describing for example, the per-

on who first reported/described the taxon in Chile, relatedomonyms, etc.; (f) its geographical distribution (i.e. whetherhe species occurs in continental Chile and/or insular Chilend/or the Chilean Antarctic territory); and (g) references (i.e.anuscripts that mention the occurrence of the taxon in thehilean territory). This additional information was compiled

rom the consulted literature as well as from our personalbservations.Classification of the species at higher taxonomic levels

ol-lowed Adl et al. (2012). These authors proposed aierarchical system of classification founded on robusthylogenetic relatedness that avoid the use of formal rankesignations such as “class”, “sub-class”, “super-order”, ororder”. The reason to do so has been motivated by utility,o avoid the common problem of a single change causing aascade of changes to the system (Adl et al. 2012). Thelassification of those taxa not included in the revision ofdl et al. (2012) was based on other authors (e.g.eisterfeld 2002a, 2002b; Haman 1988; Golemansky and

odorov 1996).

iversity analyses

We used the information compiled in the final database toalculate the total number of valid testate amoeba taxaecorded so far within the whole Chilean territory includingenera, species, infraspecific taxa and unidentified species.e then recalculated the total number of testate amoeba taxa

nly for continental Chile, insular Chile and the Chileanntarctic territory to assess the differences in diversity

mong these territories. All these calculations were alsoone sep-arately on amoebozoan and SAR testate amoebae.e then constructed a summary table for these data. In

ddition, we counted the number of species included in eachmoebozoan and SAR genus to assess their contribution tohe overall diversity. We also constructed speciesccumulation curves calculated in function of time toocument the rate of species description over the years inhile. Finally, we assessed the completeness of the

nventory of Chilean testate amoeba species richness usinghe samples collected by the first author (see databaseonstruction) as a reference sample to compute sample-size-ased rarefaction and extrapolation species rich-ness curvessee Colwell et al. 2012). The extrapolation curve wasalculated up to double the size of the reference sample.arefaction and extrapolation curves, as well as their 95%onfidence intervals (bootstrap method, 20,000eplications), were computed using R 3.1.0 and the iNEXTackage (Hsieh et al. 2014).

abitats explored and habitat preference

We investigated which habitats have been explored to studyhe Chilean testate amoebae. To do this, we looked at ouratabase and listed all the habitats reported there. Then, we

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lassified each habitat as terrestrial, aquatic and semi-quatic. Here, we also introduced a new type of semi-quatic habitat locally known as Mallín (Mallines in plural). Mallín (from the Mapudungun language = flooded place)

s a wetland that can be roughly described as a swampyrassland with min-eral soils. This type of habitat isommon in southern Chile, southern Argentina and theirdjacent islands (Schlatter and Schlatter 2004). A Mallínemains wetter than the adjacent soils in summer and isommonly flooded during winter, favoring the concentrationf organic matter and the devel-opment of wetlandegetation such as plants of the genera Acaena, Azorella,estuca and Juncus. Depending on the structure of the soil, Mallín sometimes can even harbor medium-sized nativerees (Schlatter and Schlatter 2004). While a Mallín can alsoontain some isolated Sphagnum patches, it differs from aeatland in that it has a completely different soilomposition and pH, among other physico-chemicalharacteristics. Refer to Fig. S1 for an example of a Mallín.able S1 to see some of the physicochemical properties thatharacterize and differentiate a Mallín from a peatland. Wenally counted the number of species that have beenecorded in each habitat as a measure of habitat prefer-enceor these protists, where habitat preference is the use ofome habitats over others by the organisms (Hall et al.997). This information was also explored for Amoebozoand SAR and then put in a summary table.

iogeographical aspects

To evaluate latitudinal patterns in the distributions ofestate amoeba species, we divided continental Chile into 1◦atitudinal bands (i.e. 39 latitudinal bands), registering theresence or absence of each species in each latitudinal band.he presence−absence data were used to construct a binaryatrix with columns as latitudinal bands and rows as taxa.aps of species distributions were created using data with a

patial resolution of 1◦ of latitude. Ranges were assumed toe continuous between latitudinal bands. To describe latitu-inal patterns of species, we categorized species distributionanges into three groups: (a) species with small distributions,ccurring only within two latitudinal bands; (b) speciesith small–medium ranges of distribution, ranging from

hree to 10 latitudinal bands; (c) species with medium–largeistributions, ranging from 11 to 19 latitudinal bands; andd) species with large distributions, ranging from 20 to 39atitudinal bands.

To determine if the taxa observed in Chile were character-zed by a wide or more restricted geographical distribution,e classified the species as: (a) widely distributed, if theyccurred in more than one continent; (b) South American,f they only occurred in South America; (c) Austral, if theynly occurred in the southern part of Chile and Argentina

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4

ca. 40 S to 56 S); and (d) endemics, if they only haveeen recorded in Chile.

Moreover, it has been proposed that the testate amoebapecies composition of insular Chile and the Chilean

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ntarctic territory is only a subset of that present in con-inental Chile (Smith 1985; Zapata and Matamala 1987;apata and Crespo 1990; Fernández et al. 2012). To test thisypothesis, we performed a nested analysis on the binaryatrix mentioned above. Nested analysis predicts the exis-

ence of a highly ordered system in which taxa compositionn depauperated sites tend to be simple subsets of richernes (i.e. a nested pattern) (Wright et al. 1998; Fernández015). We used the method of Atmar and Patterson (1993)nd the temperature metric (T◦) to explore the occurrence of nested pattern. The temperature metric evaluates theegree of disor-der as estimated by the temperature of ainary matrix, where T◦ = 0◦ indicates perfect nestednessnd T◦ = 100◦ indicates complete randomness. Beforealculating the degree of nestedness, the binary matrix wasorted according to the marginal row and column sums,ith common taxa placed in the upper rows and taxa-rich

atitudinal bands placed in the left-hand column (Ulrich etl. 2009; Fernández 2015). Islands and the Antarcticontinent were treated as having a direct borderline withontinental Chile. We also calculated the degree ofestedness only for Amoebozoa and then only for SAR. Thetatistical significance of these analyses was evaluated with row−column null-model (50,000 permu-tations, p < 0.05) conservative algorithm that minimizes Type I errorsGotelli 2000). Nestedness and null-model calculations asell as temperature matrices were done using R 3.1.0 and

he vegan package (Oksanen et al. 2013).

esults

nalyses on testate amoeba diversity

During the analysis of the raw database, we identified twoomonyms genera, transferred seven species to other generacombinatio nova), synonymized 83 specific and infraspe-ific taxa, and identified four invalid species (nomen nudumaxa). All these homonyms, transferred, synonymized andnvalid taxa are properly identified in the species checklistTables S2 and S3). After these procedures, the total number

f valid testate amoeba taxa recorded in Chile reached 352 nfrageneric taxa distributed in 64 genera (Table 1). From hese taxa, 24 species (17 amoebozoan and 7 SAR) are here eported for the first time for Chile (Tables S2 and S3).

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able 1. Total number of generic and infrageneric valid testate amoeba tand its three political divisions.

Chile Continental Chile

axon TA AMO SAR TA AMO S

enera 64 36 28 64 36 2pecies 255 184 71 252 181 7nfraspecific taxa 60 46 14 57 43 1nidentified species 37 27 10 30 21

e recalculated the total number of taxa for each of thehree Chilean political divisions, we found that continentalhile is much more diverse than insular Chile and thehilean Antarc-tic Territory (Table 1). In addition, when wealculated the number of taxa for Amoebozoa and SAR, weound that the first group is much more diverse than theecond, both in the whole Chilean territory and in any of itsolitical divisions (Table 1, Tables S2 and S3).

Among Amoebozoa, Difflugia and Centropyxis are theost diverse genera with 48 (18% of the total) and 41 (16%)

axa, respectively (Fig. 2a). Other genera such as Arcella (23axa, 9%), Nebela (18 taxa, 7%), Cyclopyxis (15 taxa, 6%),eleopera (14 taxa, 6%) and Argynnia (11 taxa, 4%) alsoade an important contribution to the Amoebozoa diversity

Fig. 2a). These genera together accounted for 66% of theotal amoebozoan testate amoeba diversity recorded inhile. The other 29 genera accounted for the remaining 34%f the total amoebozoan testate amoeba diversity (Fig. 2a).

Euglypha was the most diverse genus of SAR testatemoebae with 32 taxa (34% of the total) (Fig. 2b). Otherpecies-rich genera were Corythion (seven taxa, 8%), Pseu-odifflugia (seven taxa, 8%), Sphenoderia (six taxa, 7%) andrinema (11 taxa, 5%). Together these five genera representedbout 61% of the total SAR testate amoeba diversity (Fig. 2b).he other 23 genera made a low contribution to the wholeiversity as together they accounted for no more than 39% ofhe total diversity reported in Chile (Fig. 2b).

The counting of the species richness within each genuslso revealed large gaps in species identification. At least 37estate amoeba species (11% of the total) remain unidentified.he gaps in species identification are particularly important

n Amoebozoa genera such as Arcella (22%) and Phryganella40%), as well as in SAR genera such as Euglypha (13%) andseudodifflugia (57%).The assessment of the evolution of the number of species

ecorded as a function of time showed that the recorded andescribed diversity of testate amoebae in Chile has been con-inually increasing over time without leveling off (Fig. 3a).his analysis revealed an increase of over 20% (64 taxa) in

he number of new taxa reported for Chile since the year 2000

5

i.e. 15 years). This pattern was recorded for both Amoebozoa nd SAR, with increases of 21 (49 taxa) and 17% (15 taxa), espectively (Fig. 3b, c). The assessment of the completeness f the inventory of Chilean testate amoeba species richness

xa (TA), Amoebozoa taxa (AMO) and SAR taxa recorded in Chile

Insular Chile Antarctic territory

AR TA AMO SAR TA AMO SAR

8 14 12 2 13 10 31 32 28 4 23 14 94 4 4 0 0 0 09 5 5 0 2 1 1

Fig. 2. Percentage of infra-generic taxa included in each (a) amoebozoan genus and (b) SAR genus in Chile. Only the most diverse genera(those that made up over 60% of the total diversity either in Amoebozoa or SAR) are detailed.

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When analyzing the latitudinal patterns of the species, wefound that only a small fraction exhibit widespread distri-bution along continental Chile. Of the 252 testate amoebae

6

ig. 3. Species accumulation curves calculated in function of time festate amoebae.

howed a steep slope for the rarefaction curve, indicating thathe current sampling effort (i.e. the reference sample) has noteen enough to detect the whole species diversity. This anal-sis also showed a steep slope for the extrapolation curve,uggesting that Chilean testate amoeba species diversity isar from complete, even if we double the current samplingffort (Fig. 4).

abitats explored and habitat preference

A thorough analysis of the database constructed for thistudy revealed that testate amoebae were recorded from 37ifferent habitats (Table 2). Of these, 17 habitats werelassi-fied as terrestrial, 12 as aquatic, and six as semi-quatic. Most testate amoeba species were found in semi-quatic (peat-lands, 110 species) and terrestrial (rainforestoils [litter], 94 species) habitats (Table 2). Amoebozoanestate amoe-bae were commonly found in aquatic (pondediments, 93 species) and semi-aquatic (peatlands, 91

pecies) habitats, whereas SAR testate amoebae were more ften documented in terrestrial (rainforest soils [litter], 35 pecies) and semi-aquatic (peatlands, 20 species) habitats Table 2, Tables S2 and S3).

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all testate amoebae, (b) amoebozoan testate amoebae, and (c) SAR

iogeographical analyses

ig. 4. Size-based rarefaction (solid curve) and extrapolation (dot-ed curve) with 95% confidence intervals (shaded area, based on aootstrap method with 20,000 replications). The reference samples indicated by a solid black dot.

Table 2. Habitats explored to study the Chilean testate amoebae (TA), including amoebozoan testate amoebae (AMO), SAR testate amoebae,and the total number of species that have been recorded in each habitat.

Type Habitat TA AMO SAR

Terrestrial Acid mixed soils 14 6 8Arid shrublands 25 16 9Conifer forest soils (native) 9 3 6Humic soil lawns 26 16 10Humus in trunks 24 15 9Humus on trunks 20 14 6Magellanic rainforest soils 9 5 4Mosses and lichens covered by snow 9 0 9Mosses on trees (living trees) 7 3 4Mosses on trunks (dead trees) 15 9 6Muddy soils 20 15 5North Patagonian rainforest soils 19 19 0Rainforest mosses 29 21 8Rainforest soils (litter) 94 59 35Soil humus 14 11 3Soil mosses 85 61 24Sphagnum mosses 34 21 13

Aquatic Estuaries 20 19 1Forest streams 64 50 14Freshwater column 10 5 5Glacial rivers 7 4 3Hot springs 6 3 3Lake sediments 16 13 3On aquatic plants 33 27 6On freshwater algae 13 10 3Pond sediments 93 77 16River sediments 25 24 1Stream sediments 52 47 5Volcanic crater lakes 12 7 5

Semi-aquatic Mallines 27 22 5Peatlands 110 91 20Saltmarshes 10 9 1Sandy supralittoral 17 2 15Swamps 72 61 11Volcanic crater glaciers 4 0 4

Fig. 5. Latitudinal distribution patterns recorded for 252 testate amoeba species in continental Chile. (a) 102 species (40% of the total) havesmall distributions; (b) 70 species (28%) exhibit small–medium ranges of distribution; (c) 52 species (21%) have medium–large distributions;and (d) 28 (11%) species have large distributions. Each vertical band represents the distribution of a single species.

7

Fig. 6. Scanning electron microscopy and light micrographs of typical testate amoeba taxa found in Chile: (a) Alocodera cockayni (Penard, 1910): (a.1) from a peatland, TDF; and (a.2) living individual from a rainforest, SC. (b) Apodera vas Jung, 1942: (b.1−b.3) three different morphotypes that co-occur in peatlands of CC (from Zapata and Fernández 2008); and (b.4) living individual from a peatland, TDF. (c) Argynnia dentistoma (Penard, 1890): individual from a rainforest, IC (from Fernández et al. 2012). (d) A. gertrudeana Jung, 1942: (d.1) indi-vidual from a swamp, SC; and (d.2) living individual from a rainforest, CC. (e) A. schwabei Jung, 1942: from a peatland, CC. (f) Centropyxis aculeata (Ehrenberg, 1838): individual from a hot spring, SC. (g) Certesella martiali (Certes, 1889): (g.1) individual from a Mallín, CC; (g.2) living individual from a peatland, TDF; and (g.3) close-up of the neck of the previous individual, showing both the two rows of internal teeth that run along the neck (giving it a dotted appearance) and the small lateral pores that are usually present in this taxon (circles). (h) C. certesi (Penard, 1911): (h.1) individual from soil mosses, SC; (h.2) living individual from a peatland, TDF; (h.3) lateral view of the previous individ-ual, showing the internal teeth of the neck (circle 1), the internal tube that connect the two big and invaginated pores that characterized the genus

8

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pecies that have been recorded in continental Chile: (a) 102pecies (40% of the total) exhibit restricted distributions,ccurring only within two latitudinal bands (Fig. 5a); (b) 70pecies (28%) exhibit small–medium ranges of distribution,anging from three to 10 latitudinal bands (Fig. 5b); (c) 52pecies (21%) exhibit medium–large distributions, rangingrom 11 to 19 latitudinal bands (Fig. 5c); and (d) 28 (11%)pecies exhibit large distributions, ranging from 20 to 39atitudinal bands (Fig. 5d).

Furthermore, a high number of species occurring in Chilexhibit a wide geographical distribution although an impor-ant proportion also appears as unique to southern Chile. Inetail, we determined that: (a) 269 species (86% of the total)re widely distributed; (b) three species (1%) are restrictedo South America; (c) 11 species (3%) are austral; and (d)1 species (10%) are endemic to Chile. A further analysisevealed that almost all the endemic species are restricted toouth-central Chile an area included within the so-calledhilean hotspot. The endemism recorded is due to the highumber of unique amoebozoan and SAR testate amoebaaxa present in this region, which, respectively, exceeds 10nd 8%of the total number of species recorded in Chile. Theaxa that are potentially endemic to South America, toustral South America and Chile are highlighted in thedditional notes sec-tion of Tables S2 and S3. Figs 6 and 7how SEM and light micrographs of some of theseotentially endemic taxa and other typical taxa found inhile. It is noteworthy that these figures show for the first

ime some species that were previ-ously only known fromoor quality drawings or photographs. These images thusonfirm their existence.

The temperature metric confirmed that insular andntarc-tic species composition represents a subset of thatresent in continental Chile at different taxonomic criteriaor all testate amoebae, for amoebozoan taxa, and for SARaxa (Table 3). However, a few exclusive taxa have alsoeen recorded in both insular Chile and the Chileanntarctic territory (Tables S2 and S3).

iscussion

In Chile, testate amoeba diversity is very high (Table 1),ith a magnitude comparable to or even higher than in other

egions of the planet. The Chilean testate amoeba diversity

ppears two or three times higher than that of other Neotrop-cal countries, such as Peru, Colombia, Ecuador or Mexico Haman and Kohl 1994; Escobar et al. 2005; Krashevska et al.

tttt

ertesella (circle 2), and unusual internal lateral teeth (circle 3). (i) Cycloj) Cyphoderia ampulla (Ehrenberg, 1840): (j.1) individual found in a poacillifera Penard, 1890: individual from a peatland, CC. (l) D. lanceolal. 2012). (m) D. oblonga Ehrenberg, 1838: living specimen found in a prom an arid shrubland, NC. (o) Heleopera petricola Leidy, 1879: (o.1) C; and (o.2) a representative of the typical form found in a peatland, T

n a peatland, CC. (q) L. spiralis (Ehrenberg, 1840): found in a pond, SCC: northern Chile; CC: central Chile; SC: southern Chile; TDF: Tierra d

007; Bobrov and Krasilnikov 2011). The testate amoebaiversity recorded in Chile even exceeded that recorded inrgentina or China, countries that historically have experi-

nced a sampling effort comparable to that observed in ChileVucetich and Lopretto 1995; Qin et al. 2011). However,one of these countries have been systematically sampledhrough all its biomes in the way we sampled Chile. Still,hen com-pared with well-studied regions of the world,hilean testate amoeba diversity stands high. A recent

evision, (Smith et al. 2008) shown that so far 229 speciesave been recorded in North and Central America, 428 inustralia, 648 in Africa and 1031 in Europe. Thus, thehilean diversity is exceeded only by that recorded inustralia, Africa and Europe. Note, however, that we are

omparing here the diversity observed in Chile withagnitudes of diversity recorded in whole con-tinents.oreover, we do not know to what extent these comparisons

ould be valid because as stressed by Smith et al. (2008), theigher diversity recorded for some regions might be only aeflection of intensity of sampling, rather than true higheriversity. Chile, for instance, has only started to be sampledystematically in the middle of the twentieth century, whileuropean countries have been surveyed dur-ing more than

wo centuries (Foissner 1997, 1999). In turn, it is probable,or instance, that countries such as Colom-bia that arecknowledged to contain diversity hotspots for manymacroscopic) taxa (Myers et al. 2000) harbor an even largeriversity than Chile. However, a representative area of Chilelso is regarded as a biodiversity hotspot (Arroyo et al.004), and some studies have highlighted the independentrigin of its diversity (Segovia and Armesto 2015). Furtherurveys will perhaps confirm whether Chilean diversity sur-asses other under-sampled regions. In any case, the numberf species recorded in Chile has increased over the time. Ourtudy indeed suggests that the number of species recorded forhile will continue increasing, as the evolution of the speciesumber recorded as a function of time have not yet reached alateau (Fig. 3). Likewise, sample-size-based rar-efactionnd extrapolation curves show that previous studies haveverlooked a large fraction of testate amoeba speciesiversity and that many species still remains undiscoveredFig. 4).

The diversity of testate amoebae was much higher inonti-nental Chile than in the Chilean Antarctic territory andnsular Chile (Table 1). This finding is in agreement with

9

he obser-vation of several authors who have reported that he species composition found in both the Chilean Antarctic erritory and insular Chile is only a depauperated subset of hat found in

pyxis eurystoma Deflandre, 1929: individual from an estuary, CC.nd, CC; (j.2) living individual from soil mosses, SC. (k) Difflugia ta Penard, 1890: individual from a stream, IC (from Fernández et eatland, SC. (n) Euglypha strigosa (Ehrenberg, 1871): individual

individual of H. petricola major Cash, 1909 found in a rainforest, ierra del Fuego. (p) Lesquereusia modesta Rhumbler, 1895: found . Figures (d) and (e) are the first pictures ever taken for these taxa. el Fuego; IC: insular Chile.

Fig. 7. Scanning electron microscopy and light micrographs of typical testate amoeba taxa found in Chile: (a) Nebela barbata psilonata Jung, 1942: from a rainforest, IC (from Fernández et al. 2012). (b) Netzelia wailesi (Ogden, 1980): from an arid shrubland, NC (from Fernández 2015). (c) Padaungiella lageniformis (Penard, 1902): from a rainforest, IC (from Fernández et al. 2012). (d) P. wailesi (Deflandre, 1936): living specimen from soil humus, CC. (e) Plagiopyxis callida Penard, 1910: from soil mosses, CC. (f) P. labiata Penard, 1911: found in humus, CC. (g) Puytoracia jenswendti Santiba˜nez et al., 2011: (g.1) individual from a volcanic crater glacier, SC; (g.2) oral aperture; and (g.3) detail of the aboral horn or spine (from Santiba˜nez et al. 2011). (h) Scutiglypha cabrolae De Smet and Gibson, 2009: from an arid shrubland, NC (from Fernández 2015). (i) Sphenoderia rhombophora Bonnet, 1966: (i1) and (i2) show the same individual which was found in a forest dominated by Pilgerodendron uviferum. (j) S. valdiviana Chatelain et al., 2013: (j.1) individual from a rainforest, SC; (j.2) living individual from soil mosses, SC. (k) Tracheleuglypha dentata (Penard, 1890): (k.1) individual from an arid shrubland, NC; and (k.2) detail of the oral aperture. (l) Trigonopyxis arcula (Leidy, 1879): from an estuary, SC. (m) Trinema complanatum Penard, 1890: (m.1) individual from an arid shrubland, NC (from Fernández 2015); and (k.2) detail of the oral aperture. (n) Trinema lineare Penard, 1890: individual found in soil mosses,(n.1) frontal view; and (n.2) posterior view. Figure (i), is the first SEM picture ever taken for this taxon. NC: northern Chile; CC: central Chile; SC: southern Chile; TDF: Tierra del Fuego; IC: insular Chile.

10

Table 3. Degree to which species-poor communities inhabiting insular Chile and the Chilean Antarctic territory are subsets of species-richcommunities located in continental Chile. Nestedness was based on a presence−absence data matrix ordered according to the marginalrow/column sums. Below are shown the observed and expected values of the temperature metric for all testate amoebae, for testate amoebaebelonging to the Amoebozoa and for testate amoebae belonging to the SAR. Statistical inference was based on the lower and upper confidencelimits (L95% CL and U95% CL, respectively) of the null distribution of 50,000 randomized matrices.

Temperature metric

Observed Expected L95% CL U95% CL p-value

Testate amoebae 1.74 6.83 1.97 12.73 0.001AS

c Z a a t t ae o t ( At t a e i w o a

a t 1t t p fio c d p t i 1 h a o m tttde

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d C t N b 2 t o a t

11

moebozoa 0.95 5.32AR 0.82 6.27

ontinental Chile (Smith 1985; Zapata and Matamala 1987;apata and Crespo 1990; Fernández et al. 2012). Our nestednalyses underpinned this trend by showing the existence of nested pattern of species composition between these threeerritorial divisions (Table 3). However, this does not meanhat exclusive, and thus, potentially endemic species arebsent in the Antarctic and insular territories. Indeed, sev-ral species have only been recorded in these regions whilethers could not be identified to species level, suggestinghat these territories could harbor an unexplored diversityFernández et al. 2012). The low diversity recorded in thentarctic can be attributed mainly to the existence of hos-

ile environmental conditions and lack of suitable habitat forhe establishment of viable populations (Smith 1985; Zapatand Matamala 1987). However, this cause cannot be used toxplain the low diversity in insular Chile. Chilean islandsnclude habitats similar to those found in continental Chile,hich in turn are potentially suitable for the establishmentf testate amoebae (Zapata and Crespo 1990; Fernández etl. 2012).

Among the possible causes for the low insular diversityre the remoteness and the area of the islands. According tohe theory of island biogeography (MacArthur and Wilson963), taxa diversity is a decreasing function of the isola-ion of the island and an increasing function of the area ofhe island. Thus, taxa composition in insular Chile must be aauperized subset of that recorded in the continent becauserst, only good dispersers may colonize islands; and sec-nd, the area of the islands represents a small fraction of theontinent. However, while the area per se probably does notirectly determine the protist species richness of islands, itrobably determines the heterogeneity of habitat (e.g. pH,emperature and moisture ranges) and thus, the number ofnter-specific interactions that occur on islands (Hutchinson957). Besides the above prediction, the airborne dispersalypothesis (Wilkinson 2001; Lara et al. 2011; Wilkinson etl. 2012) predicts that the small species (those having a sizef below a certain threshold, such as 100 or 20 � m) areore likely to be passively dispersed over long distances

han large species. Although the mechanism proposed by his hypothe-sis could be a surrogate of other physiological raits of protists (e.g. perhaps, larger species are worse ispersers because they have more labile cysts) (Fernández t al. 2012) it arises as a

rPcLa

2.53 10.64 0.0013.12 11.52 0.001

otential cause in our study. Continental islands (i.e. islandshat lie on the continental shelf) are closer to continental Chilend indeed contain several large taxa (Fernández et al. 2012),hile oceanic islands (i.e. islands that do not sit on the con-

inental shelf) are farther from the continent and are almostree of large taxa (Zapata and Crespo 1990).

Moreover, when compared with continental Chile, theiversity of insular Chile and the Chilean Antarctic territorys abysmally low (Table 1). We suggest that this pattern is, inart, an artifact of the method used to identify the species inhese territories. There, species have been historically iden-ified with a morphological approach. This approach doesot allow discriminating between pseudocryptic and cryp-ticpecies and therefore may underestimate protist diversity.e think that the use of molecular approaches to identify

pecies in these territories would help to detect various pseu-ocryptic and cryptic species, increasing the known numberf species for insular Chile and the Chilean Antarctic terri-ory. However, by saying this we are not asserting that these of molecular approaches will show that these territoriesre equally or more diverse than continental Chile. Mostikely, diversity in these territories would still be largely aubset of that found in continental Chile (e.g. as predicted byhe theory of island biogeography). Nonetheless, the use of

olecular approaches certainly would show that theseerritories also harbor endemic species that do not co-occurn continental Chile and that they are not as poor in speciess shown by the current data.

As a general rule, we recorded higher testate amoebaiversity within the Amoebozoa than within the SAR inhile and all its political divisions (Table 1). Amoebozoan

estate amoebae mainly include large taxa (e.g. Certesella,ebela), while SAR testate amoebae are mostly representedy very small taxa (e.g. Trinema, Sphenoderia) (Meisterfeld002a, 2002b). Thus, it is not clear whether this is a realrend (e.g. Amoebozoa exhibits higher diversification rates)r only a bias introduced by the fact that very small speciesre often overlooked or hardly differentiated. In support forhe latter explanation, a recent environmental DNA survey

evealed in forest litter samples the presence of aulinellidae; a taxon of SAR largely overlooked by onventional methods in terrestrial habitats (Tarnawski and ara 2015). Likewise, detailed (SEM) morphological nalyses have shown that

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mall difference in scaling patterns can discriminate some-imes quite genetically divergent species of SAR (Wylezicht al. 2002; Chatelain et al. 2013).

Our analyses showed that Chilean testate amoebae areery diverse in semi-aquatic or terrestrial habitats such aseatlands and rainforest soils (Table 2). These habitats areuite common and abundant at mid-latitudes (Gajardo 1994;chlatter and Schlatter 2004), coinciding with areas where

here is a constant tradeoff between water and solar energynputs throughout the year (i.e. temperate areas) (Arroyo etl. 2004). The ecological conditions that prevail in thesereas certainly favor the occurrence of a high diversity, sinceater availability and mild temperatures are particularly

ritical for the survival and reproduction of testate amoebaeMeisterfeld 2002a, 2002b; Fernández 2015). In fact, theeographical dis-tribution of most species is restricted to theemperate zone, and few species extend its geographicalistribution beyond mid-latitudes (Fig. 5). Moreover, ournalyses showed that some representatives of these protistsan also occur in hot and dry habitats or cold and dryabitats such as arid shrub-lands and glaciers (Table 2).hese habitats are common at low and high latitudes

Gajardo 1994; Zapata and Matamala 1987), where there is aonstant deficit of water or solar energy inputs over the year,espectively (Arroyo et al. 2004). Because of theseharacteristics, these habitats are consid-ered suboptimal forestate amoebae (Smith 1985; Zapata and Matamala, 1987;ernández 2015). This lead us to suggest that populations

iving in these habitats are not self-sustaining and therefore,re supported by continual immigration of individualsoming from optimal habitats (temperate areas), whereopulations are actively growing and reproducing (i.e. aource-sink dynamics at biogeographical scale, seeernández 2015). The fact that most of the species thatccurs at low and high latitudes also occur at mid-latitudesupports this idea (i.e. protist species composition at low andigh latitudes are mainly a subset of that found at mid-atitudes)(Fig. 5).

Our study also revealed that one-third (33%) of testatemoeba species recorded in Chile are widely (20−38 latitu-inal bands) or relatively widely (11−19 latitudinal bands)istributed all along continental Chile while other wereeported over shorter latitudinal ranges (Fig. 4). Speciesxhibiting vast geographical distributions all along Chile areften also generalists with wide ecological tolerances toarious environmental factors, such as Arcella vulgaris orentropyxis aculeata. The first species can tolerate high saltoncentrations due to leaching from de-iced roads (Roe andatterson 2014), and can live in activated sludge (Jaromin-len et al. 2013) as well as in acidic Sphagnum mosses

nvironment (Mieczan 2007). The second occurs from alka-ine lakes (Qin et al. 2013) to Amazonian peatland poolsSwindles et al. 2014), tolerates also well organic pollution

Dorgham et al. 2013) and can survive in arid soils that are ry for much of the year (Fernández 2015). Because of their ol-erance, they have a high colonization potential; these pecies are considered as cosmopolitan.

weda

However, most widespread species have never been stud-ed with molecular methods. An exception here is Nebelaollaris, which has been found to be rather a complex ofpecies than a single entity, encompassing nowadays at leastight “morpho-phylogenetic species” (Singer et al. 2015). Its therefore possible that seemingly widespread speciesomprise several entities with more restricted distributions. similar case occurs with some relatively widespread taxa

uch as Apodera vas and Certesella spp. These taxa areonsistently distributed in southern and central Chile butheir latitudinal distribution is abruptly cut northward by theo-called ‘South American arid diagonal’, where cli-maticonditions become drier (ca. 30◦ S; southern edge of thetacama Desert). They exhibit high morphological variationithin and between populations along their latitu-dinalistributions, and morphological parameters measured showignificant discontinuities (Zapata and Fernández 2008; Fig.). This suggests that these taxa represent species com-lexes consisting of several closely related species withore restricted distributions than A. vas and Certesella spp.

ensu lato. Moreover, species exhibiting restrictedeographical distributions in Chile are both very rare andedium- to large-sized taxa (ca. 70−250 �m) that inhabit

pecific habi-tats, such as Argynnia schwabei, A.ertrudeana, Puytoracia jenswendti and Scutiglyphaabrolae (Figs 6 and 7). The first two species are only foundithin habitats included in the southern rainforest biome

Zapata 2005; Fernández and Zapata 2011), the third haseen found only on the surface of a glacier (Santiba˜nez etl. 2011) and the fourth is only known from an isolated highltitude volcanic lake in the Andes Range (De Smet andibson 2009) and extremely arid soils of the southern edgef the Atacama Desert (Fernández 2015). Most of thepecies with restricted geographical distributions are alsondemic to Chile and their distributions are within thehilean biodiversity hotspot. The value of endemism

ecorded so far for Chile reaches close to 10% of the totaliversity. Some researchers believe that a value close to0%is sufficient for an area to qualify as a geographicalrovince, at least for macroscopic organisms (Briggs andowen 2012). Therefore, the Chilean biodiversity hotspotould potentially host a unique micro-eukaryotic diversitynd represent an area of evolutionary innovations or refugeshere an older and still understudied microbiota persists.e trust that future molecular-based studies and

hylogeographic analyzes will enable us to test thisypothesis.

onclusions

The Chilean territory represents an area of high diversityor testate amoebae. In this country, testate amoeba diversityas exponentially increased in recent years and this trend

12

ill continue over time. This conjecture is supported by themerging use of molecular techniques, which promise toetect a cryptic diversity largely overlooked by traditionalpproaches. Testate amoebae also comprise a widespread

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roup in Chile that occurs in many habitats, though they areore diverse in peatlands and rainforest soils. However, this

attern may be an artifact of uneven sampling effort, since inhile the latter habitats have been more sampled than others.his calls for studying other less explored habitats (e.g. hotnd cold deserts, glaciers) to expand our view on its ecologyhus allowing us to use them as bioindicators of the past andresent-day environmental conditions in this region of thelanet.

Testate amoebae are not randomly distributed in Chile.pecies composition of Insular Chile and the Chilean Antarc-

ic territory is mainly a depauperated subset of that found inontinental Chile. This nested pattern of species composi-ion has been also found for several metazoans and has beenxplained in terms of the theory of island biogeography. Weuggest that the occurrence of this pattern also is driven byhe same mechanisms proposed by this theory (i.e. selectivextinction and selective colonization).

Moreover, in continental Chile only generalist species areidely distributed (ca. 11% of the total), occurring from

em-perate to extremely hot and cold biomes. In contrast,he remaining 89% of the species have a much moreestricted distribution, occurring mainly in the temperateone where there is a constant tradeoff between water andild temper-atures inputs over the year. At least the 10% of

hese species are potentially endemic to Chile and occurxclusively in tem-perate biomes of the so-called Chileaniodiversity hotspot. These diversity patterns have beenepeatedly found for Chilean metazoans and have beenxplained in terms of ecological, historical and evolutionaryauses. For exam-ple, the lift of the Andes and theleistocene glaciations changed the climate in this region,reating hot and cold cli-mates at high and low latitudes,espectively. These changes forced the species to flee intoid-latitudes (temperate areas), where they undergone

volutionary events that resulted in new species. Then, sinceot and cold climate conditions still prevail at low and highatitudes, very few species (mainly generalist species) haveeen able to re-colonize these areas (Arroyo et al. 2004). Weuggest that these processes also determined the spatialistribution of these protists in conti-nental Chile.

Finally, the occurrence of the aforementioned diversity pat-erns suggests that, in Chile, testate amoebae and metazoansxhibit analogous diversity patterns driven by similar under-ying mechanisms. Future biogeographical, macroecologicalnd phylogeographic analyses may validate this idea.

cknowledgements

L.D.F. was supported by CONICYT (doctoral scholar-hips No. 21110037 and 78130011) and the Universidad

e Concepción (Dirección de Postgrado). Field expedi-ions conducted by L.D.F. in Chile were funded by theollowing institutions (in alphabetic order): Ministerio del

edio Ambiente del Gobierno de Chile (FPA project N◦

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1-007-08), the Wildlife Conservation Society (Karukinkarant No 2012), Universidad de Los Lagos (project No. 2008:oraminíferos de marismas salobres del Sur de Chile), andniversity of Neuchâtel (mobility grant, IDPOB). We thankranziska Sorge and Ildiko Szelecz for helping with Ger-an translations; Pamela Santibanez and Centro de Estudiosientíficos-Chile for sharing with us SEM pictures of P.enswendti; Lorena and Mónica from Naturalis (FCNYM,NLP-Argentina); Julio Pascual (Biblioteca IGME, Spain)

or sharing specialized literature; and two anonymous refer-es for their helpful comments. Special thanks to Krzysztofiackowski for authorizing the use of figures previously pub-

ished in Acta Protozool.; Jaime Zapata for providing hisrivate collection of permanent slides; Ralf Meisterfeld anduri Mazei for kindly reviewing the taxonomic information inur database; and Fabiola Barrientos-Loebel for performinghe artwork of Figs 5 and 6.

ppendix A. Supplementary data

Supplementary data associated with this article can be ound, in the online version, at http://dx.doi.org/10.1016/.ejop.2015.07.001.

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